Optimization strategies of cell culture processes aim at maximizing the longevity of cell culture (Bibila, T. A., and Robinson, D. K., Biotechnol. Prog. 11 (1995) 1-13). The final integrated number of viable cells over cultivation time is often used as a measure of cultivation success and is positively correlated with product formation. As used herein, this integral is defined as CTI (CTI=Cell density Time Integral).
Lactate is a major waste product formed during the cultivation of mammalian cells. Under typical culture conditions, the cells consume glucose in great excess and metabolize it mainly to lactate. The accumulation of lactate affects cell growth, CTI and protein production adversely as a result of pH and/or pH adjustment by alkali (Chang, Y. H. D., et al., Biotechnol. Bioeng. 47 (1995) 319-326); Omasa, T., et al., Biotechnol. Bioeng. 39 (1992) 556-565 and Chen, K., et al., Biotechnol. Bioeng. 72 (2001) 55-62).
There have been a lot of attempts to reduce lactate formation. It was suggested by Glacken, M. W., et al., (Biotechnol. Bioeng. 28 (1986) 1376-1389); Hu, W. S., et al., (Dev. Biol. Stand. 66(1987) 279-290); and Xie, L., and Wang, D. I. C., Cytotechnology 15 (1994) 17-29) to grow mammalian cells at low glucose concentrations with dynamic controlled feeding with glucose. The idea was to achieve a metabolic shift from high glucose/lactate flux to a low glucose/lactate flux. However, such methods require adaptations of the cells and need carefully designed control mechanisms of feeding. They are therefore complicated and difficult to perform (U.S. Pat. No. 6,156,570).
Other methods for reducing lactate formation are based on genetic engineering means. One method is described by Chen, K., et al., Biotechnol. Bioeng. 72 (2001) 55-62) suggesting manipulation of the metabolic pathway for lactate in the mammalian cells by inactivation of at least one copy of lactate dehydrogenase genes in the cells. Another method is described by Irani, N., et al., (J. Biotechnol. 66 (1999) 238-246), which introduces a pyruvate carboxylase gene into the host cell genome. It is assumed that the conversion of pyruvate to lactate is reduced and therefore the longevity of the cell culture is improved.
The addition of ferric citrate as a substituent for transferring in serum-free media for the cultivation of mammalian cells has been known for a long time (cf., e.g., Toyoda, K., and Inouye, K., Agric. Biol. Chem. 55 (1991) 1631-1633; Franek, F., and Dolnikova, J., Cytotechnology 7 (1991) 33-38; Kovar, J., and Franek, F., Exp. Cell Res. 182 (1989) 358-369; Schneider, Y. J., J. Immunol. Meth. 116 (1989) 65-77; and Kovar, J., Hybridoma 7 (1988) 255-263).